商业酵母在葡萄酒工业化生产中的定殖情况分析

doi:10.3864/j.issn.0578-1752.2021.09.016

摘要/Abstract

摘要：

【目的】揭示商业活性干酵母（active dry yeast,ADY）在葡萄酒工业生产中的定殖情况,及其与我国本土酿酒酵母菌株的遗传关系,为我国本土优良酿酒酵母的选育提供理论依据,同时为葡萄酒生产中ADY的使用提供参考。【方法】以宁夏贺兰山东麓葡萄酒产区的‘赤霞珠’葡萄为原料,分别接种BDX、XR、FR和FX10四种ADY发酵,于接种后的1、3和5 d取样。利用Interdelta和SSR技术对酿酒酵母进行菌株区分,分析商业ADY在不同发酵阶段的数量、比例以跟踪其定殖能力。利用PopGen32软件分析其遗传多样性相关指数,采用NTsys2.10e软件揭示商业酵母与宁夏本土酵母的遗传关系。【结果】4种ADY共显示出6种Interdelta指纹图谱即6种基因型：XR和FR均由2种株系组成,BDX和FX10均由1种基因型组成。对于本研究选取的9个微卫星位点而言,每种ADY均显示出1种基因型。4个发酵中共分离到225个酿酒酵母单菌落,Interdelta分析显示42种基因型,其中本土基因型为36种,菌株变异程度为中等16%（36/225）;SSR分析显示20种基因型,其中本土基因型为16种,处于中等遗传多样性水平。9个微卫星位点共检测出75个等位基因,平均每个位点的等位基因数为8.3333个,观测杂合度（Ho）在0.2000—0.5000,PIC在0.6339—0.8620,平均值为0.7614,均为高度多态性位点。接种BDX的发酵中本土基因型最丰富（11种Interdelta类型和8种SSR类型）,接种FR的发酵次之（11种Interdelta类型和6种SSR类型）。商业ADY没有在发酵的各个阶段占据主导地位,对于不同的发酵阶段而言,优势酿酒酵母的基因型也存在差异。Interdelta分析和SSR分析均显示FR不是相应发酵中的优势菌株。BDX虽然存在于整个发酵过程中,但只在接种后的3 d占主导地位。接种XR和FX10的发酵中,Interdelta分析法显示它们不是发酵中的主导菌株,而SSR分析显示它们均是相应发酵中的主导菌株。本土酿酒酵母基因型β（SSR类型为BDX-7）、基因型γ（SSR类型为BDX-6）、基因型A（SSR类型为XR）、基因型a（SSR类型为FX10）、基因型b（SSR类型为FX10）、基因型bb（SSR类型为FR-2）和基因型ee（SSR类型为FR-4）等在相应的发酵中均表现出较强的竞争力。聚类分析表明,分离自相同发酵中的酿酒酵母菌株间遗传差异性较大。【结论】葡萄酒工业生产中本土酿酒酵母基因型丰富,发酵是由本土酵母与商业酵母共同参与完成的,且二者在发酵中相互竞争,数量动态演替。

关键词: 活性干酵母, 接种发酵, 菌株区分, 葡萄酒, 微卫星

Abstract:

【Objective】The purpose of this study was explore the implantation and persistence of commercial active dry yeast (ADY) during industrial wine fermentations, and their competitive relationship between Chinese indigenous Saccharomyces cerevisiae during fermentation process, so as to provide the theoretical basis for the breeding of indigenous S. cerevisiae strains and provide the reference for the use of ADY in wine production. 【Method】Industrial wine fermentations were carried out at wineries in Eastern Foot of Helan Mountain in Ningxia. Four vats of Cabernet Sauvignon gape must were inoculated with BDX, XR, FR and FX10, respectively. Samples were collected and analyzed at 1 d, 3 d and 5 d after the inoculation. Interdelta and SSR analysis were used to investigate the genotypes of different S. cerevisiae strains. Therefore, the number and proportion of S. cerevisiae strains in different fermentation stages were analyzed, and the colonization ability of commercial ADY was tracked. Genetic diversity parameters were calculated by PopGen32 software. The genetic correlation between commercial yeast and Ningxia indigenous yeast was revealed by NTsys2.10e software. 【Result】Interdelta fingerprint showed 6 kinds of fingerprints, namely 6 genotypes. And XR and FR showed more than one genotype; BDX and FX10 showed one genotype, respectively. SSR analysis showed that there was one genotype in each ADY for the 9 locus. 225 S. cerevisiae isolates were isolated from the 4 inoculated fermentations. Interdelta fingerprint showed 42 genotypes, of which 36 genotypes were indigenous strains. The degree of variability (16%, 42/225) was intermediate. SSR analysis showed 20 genotypes, of which 16 genotypes were indigenous strains. The analyzed 9 microsatellite prime pairs generated a total of 75 polymorphic bands, 8.3333 alleles for per locus. The heterozygosity observed was 0.2000-0.5000. The polymorphism information contents (PIC) of all strains at 9 loci were 0.6339-0.8620, suggesting that the 9 SSR loci were hypervariable. The indigenous genotypes were the most abundant in the fermentation inoculated with BDX (11 Interdelta types and 8 SSR types), followed by FR (11 Interdelta types and 6 SSR types). ADY did not dominate all three stages. Moreover, the genotypes of the dominant strains were also different for different stages in the same fermentation. Interdelta and SSR analysis showed FR was not dominant in the corresponding fermentation. Although BDX existed in the whole fermentation process, it was only dominant at d 3 after the inoculation. In the fermentation inoculated with XR and FX10, Interdelta analysis showed that they were not the dominant strains, while SSR analysis showed that they were the dominant strains in the corresponding fermentations, respectively. Indigenous strains of genotype β (SSR genotype BDX-7), genotype γ (SSR genotype BDX-6), genotype A (SSR genotype XR), genotype a (SSR genotype FX10), genotype b (SSR genotype FX10), genotype bb (SSR genotype FR-2) and genotype ee (SSR genotype FR-4) showed strong competitiveness in the corresponding fermentations. Cluster analysis showed that the genetic diversity among the S. cerevisiae strains isolated from the same fermentation was large. 【Conclusion】The genotypes of indigenous S. cerevisiae strains in the industrial wine fermentations were rich. The inoculated fermentations were completed by both indigenous strains and commercial ADY, and they competed with each other in the same fermentations and showed dynamic succession of different strains.

Key words: ADY, inoculated fermentation, strain typing, wine, SSR

孙悦,杨慧敏,何荣荣,张军翔. 商业酵母在葡萄酒工业化生产中的定殖情况分析[J]. 中国农业科学, 2021, 54(9): 2006-2016.

SUN Yue,YANG HuiMin,HE RongRong,ZHANG JunXiang. Implantation and Persistence of Inoculated Active Dry Yeast in Industrial Wine Fermentations[J]. Scientia Agricultura Sinica, 2021, 54(9): 2006-2016.

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位点 Locus	染色体 Chromosome	重复序列 Repetitive sequences	引物序列 Primer sequence (5′-3′)	参考文献 Reference
SCAAT1	XIII	TTA	F: AAAGCGTAAGCAATGGTGTAGATACTT R: CAAGCCTCTTCAAGCATGACCTTT	[15-18]
YPL009C	XVI	CTT	F: AACCCATTGACCTCGTTACTATCGT R: TTCGATGGCTCTGATAACTCCATTC	[15,17]
C4	XV	TAA	F: AGGAGAAAAATGCTGTTTATTCTGACC R: TTTTCCTCCGGGACGTGAAATA	[15,17-18]
C5	Ⅵ	GT	F: TGACACAATAGCAATGGCCTTCA R: GCAAGCGACTAGAACAACAATCACA	[15,17-18]
C8	VII	TAA	F: CAGGTCGTTCTAACGTTGGTAAAATG R: GCTGTTGCTGTTGGTAGCATTACTGT	[15,18]
C11	X	GT	F: TGCGCAGCTTAGTATGACCA R: GATGGGCTTTCACTCCACTT	[16-18]
C12	XII	CAA	F: GAGGAGCTTACTTAAGAGCATGCGTTC R: GTGTCTTAAACCTATATTCGGATTGTGCCTGCT	[15]
SCAAT3	IV	AAT	F: TGGGAGGAGGGAAATGGACAG R: TTCAGTTACCCGCACAATCTA	[15,18]
SCYOR267C	XV	TGT	F: TACTAACGTCAACACTGCTGCCAA R: GGATCTACTTGCAGTATACGGG	[15,17]

ADY	SCAAT1	YPL009C	C4	C5	C8	C11	C12	SCAAT3	SCYOR267C
BDX	208	278	245	159	133	282	120	249	286
BDX	208	305	257	169	142	282	123	264	289
XR	205	290	254	139	136	282	120	249	321
XR	215	302	263	155	146	286	123	249	321
FX10	208	299	254	113	133	290	120	267	321
FX10	208	299	254	113	133	290	120	267	321
FR	199	299	257	151	123	276	123	244	275
FR	214	299	279	161	133	288	123	244	275

位点 Locus	等位基因数 Alleles	最长片段大小 Max. allele (bp)	最短片段大小 Min. allele (bp)	观测杂合度 Heterozygosity observed (Ho)	期望杂合度 Heterozygosity expected (He)	多态信息含量 Polymorphism information contents (PIC)
SCAAT1	9	252	174	0.3125	0.7601	0.7155
YPL009C	7	305	275	0.4375	0.7944	0.7337
C4	10	303	245	0.4667	0.8598	0.8124
C5	11	172	113	0.2500	0.8669	0.8224
C8	5	146	124	0.2500	0.7802	0.7132
C11	10	290	146	0.4375	0.9032	0.8620
C12	8	159	119	0.5000	0.6956	0.6339
SCAAT3	7	268	243	0.2000	0.8460	0.7934
SCYOR267C	8	321	275	0.3125	0.8206	0.7662